Objective

Recent improvements in the depletion of high & medium abundant proteins and development of tissue-enhanced fluid proteomics have increased the breadth and depth of plasma proteome coverage. We have now combined these methods to obtain unparalleled coverage of the IPF plasma proteome.

Method

Longitudinal plasma samples from 6 individuals with IPF receiving Pirfenidone treatment & five control individuals were depleted on an IgY14-Supermix column. Unbound proteins were digested using trypsin and resultant peptides labelled with isobaric Tandem Mass Tags^®(TMT^®). In parallel, four aliquots of IPF lung tissue digest were labelled with TMT^®.  Labelled samples were mixed to produce 5 analytical 10plex samples, each comprising 6 x 2 units of fluid proteomes and 1, 4, 6 & 10 units of tissue proteome, the latter serving as both a trigger and calibration curve. Each 10plex sample was fractionated by high pH reverse-phase chromatography yielding 30 fractions. Each fraction was analysed using a 2 hour gradient on an Orbitrap^®Fusion Tribrid^®mass spectrometer with in-line uHPLC and processed using Proteome Discoverer v1.4 and Proteome Sciences’ in-house bioinformatics workflows. To assess the impact of the tissue trigger we also performed a single 10plex analysis using samples that underwent identical depletion, TMT^®labelling and mass spectrometry but without addition of a tissue trigger.

Results

Mean protein recovery from IgY14-Supermix depletion was 96µg(0.8%) from a 12.5mg load, indicating ~99% depletion. SDS-PAGE confirmed depletion and consistent increases in proteome complexity with high intra-patient reproducibility. Table 1 summarises features detected in LC-MS/MS. Whilst the number of MS/MS spectra collected were similar by both methods, remarkably, the addition of the tissue trigger led, on average, to the quantification of twice as many proteins in plasma. TMTcalibrator^TMsubstantially enhanced or enabled detection of plasma proteins related to IPF biology, including collagen I and IV fragments, integrins (α_v, α₆, β₁and β₆) and TGFβ (1 and 2). TGFβ₂ and integrin β₆were only identified with TMTcalibrator^TM.

Conclusion

Combining IgY14-Supermix depletion with TMTcalibrator^TMtissue-enhanced plasma proteomics has substantially increased coverage of the IPF plasma proteome. Downstream mining and analysis of this large dataset will reveal novel insights into IPF biology, incl. mechanisms regulating responses to treatment. This will enable selection of potential pharmacodynamic biomarkers for downstream validation in larger cohorts.